Evidence for importance of oxidative-nitrosamine stress in peripheral diabetic neuropathy (PDN) is emerging, but the mechanisms are not well understood. Up regulation of Na? exchanger-1 (NHE-1), previously demonstrated in several tissue-sites for diabetes complications, causes an increase in cytosolic ph and related activation of the upper part of glycolysis. The overall hypothesis of this proposal is that NHE-1 plays an important role in PDN, because NHE-1-driven activation of the upper part of glycolysis, under conditions of diabetes-induced inhibition (endothelial cells) or insufficient activation (Schwann cells, SC) of glyceraldehyde- 3-phosphate dehydrogenate, underlies diversion of the excessive glycolytic flux towards formation of a-glycerophosphate and methylglyoxal, with resulting activation of two major free radical-generating mechanisms i.e., non-enzymatic glycation and NAD(P)H oxidase. In support of this hypothesis, our preliminary studies have shown that 1) NHE-1 is abundantly expressed in rat and mouse peripheral nerve, rat DRG neurons, and human SC; 2) HSC basal ph and NHE-1 expression and activity increase in response to high glucose; 3) the upper part of glycolysis is activated in the diabetic peripheral nerve; 4) the specific NHE-1 inhibitor cariporide, at least, partially prevents nerve conduction deficits, sensory neuropathy, neurovascular dysfunction, and metabolic imbalances in STZ-diabetic rats; 5) STZ-diabetic NHE-1 mice develop less severe PDN than STZ-diabetic wild-type mice. The OBJECTIVE of this proposal is to evaluate the role of NHE-1 in PDN in animal models of Type 1 and Type 2 diabetes. The specific aims are 1) elucidate if NHE-1 inhibition reverses PDN in STZ- diabetic and ZDF rats; 2) determine the roles of HIF-1a and aldosterone in diabetes- and high glucose-induced NHE-1 overexpression; and 3) evaluate the contribution of NHE-1 to oxidative-nitrosative stress in peripheral nerve, vasa nervorum, spinal cord, and DRG neurons of diabetic rats and high glucose-exposed cultured HSC and co-cultured rat SC and DRG neurons. The project combines physiological, behavioral, biochemical, immunohistochemical, and structural studies in animal models with molecular studies in cultured HSC and co- cultured rat SC and DRG neurons. The findings will generate new information on the role for NHE-1 in PDN in two types of diabetes and may provide rationale for development of NHE-1 inhibitors and NHE-1 inhibitor- containing drug combinations. PUBLIC HEALTH RELEVANCE: Peripheral diabetic neuropathy (PDN) is the most devastating complication of diabetes mellitus, and a leading cause of foot amputation. Evidence for importance of free radicals and oxidants in PDN is emerging from both animal and human studies, but the mechanisms are not well understood. Upregulation of Na? exchanger-1 (NHE-1), previously demonstrated in several tissues of diabetic animals, causes an increase in cytosolic ph and related activation of the upper part of glycolysis. The overall hypothesis of this proposal is that NHE-1 plays an important role in PDN, because NHE-1-driven activation of the upper part of glycolysis, under conditions of diabetes-induced inhibition (endothelial cells) or insufficient activation (Schwann cells, SC) of glyceraldehyde-3-phosphate dehydrogenase, underlies diversion of the excessive glycolytic flux towards two major free radical-generating pathways i.e., non-enzymatic glycation and NAD (P) H oxidase. The hypothesis is supported by our preliminary data demonstrating that 1) NHE-1 is abundantly expressed in rat and mouse peripheral nerve, rat DRG neurons, and human SC; 2) HSC basal ph and NHE-1 expression and activity are increased by high glucose; 3) the upper part of glycolysis is activated in the diabetic nerve; 4) the specific NHE- 1 inhibitor cariporide, at least, partially prevents PDN in STZ-diabetic rats; 5) diabetic NHE-1 mice develop less severe PDN than diabetic mice with normal NHE-1 content. The OBJECTIVE of this proposal is to evaluate the role of NHE-1 in PDN in animal models of Type 1 and Type 2 diabetes. The SPECIFIC AIMS are 1) elucidate if NHE-1 inhibition reverses PDN in STZ-diabetic and ZDF rats; 2) evaluate the mechanisms of diabetes-induced NHE-1 over expression; and 3) assess the contribution of NHE-1 to oxidative-nitrosative stress in peripheral nervous system of diabetic rats and high glucose-exposed cultured HSC and co-cultured rat SC and DRG neurons. The project combines physiological, behavioral, biochemical, immunohistochemical, and structural studies in animal's models with molecular studies in cell cultures. The findings will generate new information on the role for NHE-1 in PDN in two types of diabetes and may provide rationale for development of NHE-1 inhibitors and NHE-1 inhibitor-containing drug combinations for prevention and treatment of PDN.